Synthetic catechol derivatives, method for production and use thereof

ABSTRACT

Catechol derivatives of general formula (I)                    
     in which R 1  denotes O-acyl and R 2  represents amino acid residues in the 3- and/or 4-position function as siderophores and/or as biological chelating agents for iron in gram-negative bacteria. Conjugates with antibiotics improve penetration into bacterial cells, thereby increasing antibacterial efficacy of the cells.

The present invention relates to new synthetic catechol derivatives, inwhich aromatic azomethine-carboxylic acids, benzhydrazones, amino acids,aminobenzoic acids or dipeptides, pyrrolidine- or oxazolidine-carboxylicacids, or formylcarboxymethyloximes function as structural elements, andrelates to conjugates thereof with active ingredients, particularlyantibiotics.

It is known that certain catechol structures play an essential role asiron-complexing structural elements in natural siderophores (“IronTransport in Microbes, Plants and Animals”, Eds.: Winkelmann, G., vanHelm, D., Neilands, J. B., V. Ch.—Verlagsgesellschaft Weinheim, 1987),e.g. enterobactin, which is a siderophore for E. coli and otherbacterial strains, is a trimer of N-(2,3-dihydroxybenzoyl)-L-serine. Themonomer is also effective as a siderophore (Hantke, K., FEMS Microbiol.Lett. 67 (1990), 5). N-(2,3-dihydroxybenzoyl)glycine has been found tobe a siderophore for B. subtilis (Ito, T., Neilands, J. B., J. Amer.Chem. Soc. 80 (1958), 4645). Some catechol-substituted amino acidderivatives have already been produced synthetically, e.g.N-(2.3-dihydroxy-benzoyl)-L-threonine (Kanai, F., Kaneko, T., Morishima,H., Isshiki, K., Taketa. T., Takeuchi, T., Umezawa, H., J. Antibiot. 38(1985), 39), N², N⁶-bis-(2,3-dihydroxybenzoyl)-L-lysine (Corbin, J. L.,Bulen, W. A., Biochemistry 8 (1969), 757; McKee, J. A., Sharma, S. K.,Miller, M. J., Bioconjugate Chem. 2 (1991) 281), andN²,N⁶-bis-(2,3-dihydroxybenzoyl)-lysyl-N⁶-(2,3-dihydroxybenzoyl)lysine(Chimiak, A., Neilands, J. B., Structure and Bonding 58, (1984), 89). Itis also known that certain glyoxylic acid benzhydrazones, oxanilic acidderivatives, etc., can serve as siderophores for different bacterialstrains (Reissbrodt, R., Heinishc, L., Möllmann, U., Rabsch, W.,Ulbricht, H., BioMetals 6 (1993), 155). Somedihydroxybenzylidene-aminobenzoic acids have already been described inthe literature, but without any mention of their efficacy assiderophores (Takita, H., Noda, S., Inada. K., Mukaida, Y. S., Toji. M.K., Kobayashi, H., DE 3 414 049 (1984); H. Wolf, Monatsh. Chem. 31(1910), 903).

Although various catechol compounds have been bonded to β-lactams, bymeans of which an increase in the antibacterial efficacy of theseantibiotics has been achieved due to their transfer into the bacterialcell via bacterial transport routes for iron (e.g. Arisawa, M., Sekine,Y., Shimizu, S., Takano, H., Angehrn, P., Then, R. L., Antimicrob.Agents Chemother. 35 (1991), 653), there is a great need for other newsynthetic siderophores with improved pharmacological andpharmaco-kinetic properties, which are suitable for forming conjugateswith antibiotics.

On the other hand, as chelating agents for iron, siderophores arepotentially capable of influencing the biological metabolism of iron,and diseases associated therewith, in various ways. Thus the siderophoredesferrioxamine (desferal) is successfully used in diseases which arecaused by an excess of iron (e.g. thalassaemia).

The underlying object of the present invention is to discover newsynthetic catechol derivatives which comprise aromaticazomethine-carboxylic acids, benzhydrazones, amino acids, aminobenzoicacids or dipeptides, pyrrolidine- or oxazolidine-carboxylic acids, andformylcarboxymethyloximes as basic structures, which can function assiderophores and/or as biological chelating agents for iron, and whichin the form of their conjugates with active ingredients, e.g.antibiotics, effect improved penetration of these compounds intobacterial cells and thereby increase the antibacterial efficacy thereof,and which make it possible to combat penetration-related resistance toantibiotics in bacterial infections in an improved manner.

The compounds according to the invention are effective as siderophoresfor gram-negative bacteria, i.e. they can supply bacteria with ironions, and, in die form of their conjugates with active ingredients, e.g.with antibiotics (as “siderophore-antibiotic conjugates”), can transferthese compounds into the bacterial cell via iron transport routes andcan thereby improve or even extend the efficacy thereof.

Moreover, the compounds according to the invention are more effectiveand can be produced more easily than previously known compounds, and indie form of their conjugates with active ingredients make it possible tocombat penetration-related resistance to antibiotics in bacterialinfections in an improved manner. Furthermore, the present inventionprovides new chelating agents for iron, which can influence thebiological metabolism of iron and which can thus influence diseasesassociated therewith in various ways.

New synthetic catechol derivatives are provided, of general formula I

wherein the R¹ radicals are identical to or independent of each otherand denote OH and/or Oacyl, and R² represents the following groups inthe 3- and/or 4-position:

a. aromatic azomethine-carboxylic acid residues and/orazobenzene-carboxylic acid residues:

X=CM, N, CH=CH-CH

Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferablyNa, K), an ammonium ion or a substituted ammonium ion, or

Y=an active ingredient residue which contains an OH or NH group,

R³=one or two Oacyl radicals when R¹=OH or Oacyl, or

R³=H when R¹=Oacyl, or

R¹⁵=radicals which, identically to or independently of each other,represent H and/or Oacyl, or

Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferablyNa, K), an ammonium ion or a substituted ammonium ion, or

Y=an active ingredient residue which contains an OH or NH group,

R³=radicals which, identically to or independently of each other, denoteH, OH, Oacyl,

b. benzhydrazone radicals:

R¹⁵=radicals which, identically to or independently of each other,denote H, OH, Oacyl,

R⁴ and/or R⁵ is H or COY, wherein

Y=OA, with A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferablyNa, K), an ammonium ion or a substituted ammonium ion, or

Y=an active ingredient residue which contains an OH or NH group,

c. aminobenzoic acid residues

Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferablyNa, K), an ammonium ion or a substituted ammonium ion, or

Y=an active ingredient residue which contains an OH or NH group,

R¹⁹=H, alkyl,

R²⁰=H, alkyl, halogen, OH, Oalkyl, Oacyl, or

R¹⁹ and R²¹, identically to or independently of each other, each denoteH, OH, Oacyl or Oalkyl in the 2,3- and/or 3,4-position

d. amino acid residues:

Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferablyNa, K), an ammonium ion or a substituted ammonium ion, or

Y=an active ingredient residue which contains an OH or NH group,

R⁶=alkyl, hydroxyalkyl (comprising C₁-C₅ when R¹=Oacyl and C₃-C₅ whenR¹=OH), or alkoxyalkyl, acyloxyalkyl, arylalkoxyalkyl, or

R¹⁵ represents, identically to or independently of each other, H, OH,Oacyl,

n is an integer between 1 and 5 when R¹ is Oacyl and R¹⁵ is H and/orOacyl, or

n is an integer between 1 and 3 when R¹ is OH and R¹⁵ is H and/or OH, or

R¹⁵=radicals which, identically to or independently of each other,denote H, OH, Oacyl, n₁ and n₂ represent an integer between 1 and 5,

e. pyrrolidine- and/or oxazolidine-carboxylic acid residues

Z=O, CH₂,

R¹⁶ and R¹⁷, independently of each other, denote H, alkyl or aryl,

Y=OA where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferablyNa, K), an ammonium ion or a substituted ammonium ion, or

Y=an active ingredient residue which contains an NH or OH group,

f. formyl-O-carboxymethyloximes

R²=CH═NOCH₂COY, where

Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferablyNa, K), an ammonium ion or a substituted ammonium ion, or

Y=an active ingredient residue which contains an OH or NH group.

In the above formulae and hereinafter, the term “acyl” denotes astraight-chain or branched C₁-C₆ alkanoyl or a straight-chain orbranched C₁-C₆ alkoxy-carbonyl. A straight-chain or branched alkyl and astraight-chain or branched alkoxy, also in compound words such asalkoxyalkyl or acyloxyalkyl, denote a straight-chain or branched C₁-C₈alkyl or -alkoxy in particular. Aryl denotes phenyl and substitutedphenyl in particular, such as a phenyl which is substituted by astraight-chain or branched alkyl, by a halogen, particularly Cl or F, bya straight-chain or branched alkoxy, hydroxy or carboxy, or by astraight-chain or branched alkoxycarbonyl, by a halogen-substitutedalkyl or a substituted phenyl, and aralkyl denotes phenylmethyl and 1-or 2-phenylethyl in particular. The cited radicals R³, R⁵, R¹⁵, R²⁰ andCOY may be situated in all possible positions. A substituted ammoniumion is an ammonium ion which is substituted by an alkyl, for example.

The term “active ingredient residue” denotes the residue of any suitableantibacterial active ingredient comprising a free NH or OH group, forexample, wherein the active ingredient is esterified or converted to anamide with the catechol radical via this NH or OH group. The bondbetween the catechol derivative and the antibiotic can be formed eitherdirectly or via customary linker groups, e.g. aminocarboxylic acids,hydroxycarboxylic acids, diamines or diols. The term “antibiotic” is tobe understood, for example, as a corresponding β-lactam containing an NHor OH group, e.g. a cephalosporin, e.g. cephalexin, cephadroxil orclaforan, or a penicillin, e.g. ampicillin or amoxicillin, or atetracycline derivative, e.g. an aminodioxycycline, or an antibiotic ofdie aminoglycoside, macrolide, quinolone or carbapenem type.

If asymmetric C atoms are present, the invention likewise relates to thecorresponding D- and L- forms, enantiomers and diastereomers, and toracemates and mixtures of enantiomers and diastereomers.

The compounds according to the invention can be prepared for example, by

a. the reaction of catechol-substituted benzaldehydes (formula I, whereR²=CHO), in a suitable solvent such as ethanol or toluene, with a watertrap or with water-bonding means such as a molecular sieve in a soxhletattachment, at reaction temperatures between +50° C. and +120° C. andgenerally at the boiling point of the solvent, with correspondingaminobenzoic acids to form aromatic azomethine-carboxylic acids (formulaI, where R²=R⁷ or R⁸),

or by

b. the reaction of catechol-substituted benzhydrazides (formula I, whereR²=CONHNH₂), in a suitable solvent such as water, ethanol or aceticacid, at temperatures between +10° C. and +120° C. and preferably at theboiling point of the solvent, with corresponding formylbenzoic acids orwith phenylglyoxylic acids to form corresponding benzhydrazones (formulaI, where R²=R⁹),

or by

c. the reaction of di(acyloxy)benzoyl chlorides (formula I, whereR¹=OCOCH₃ and R²=COCl, for example) with aminobenzoic acids or estersthereof, in a suitable solvent such as tetrahydrofuran together with atertiary amine e.g. triethylamine, at a temperature between −30° C. and+20° C., or in aqueous sodium bicarbonate solution at 0° C. to 10° C. toform N-[di(acyloxy)benzoyl] aminobenzoic acids or esters, and thelast-mentioned esters are optionally converted into the free acids(formula I, where R²=R¹⁰),

or by

d. the reaction of 2,3-di(benzyloxy)benzoyl chloride (formula I, whereR¹=OCH₂C₆H₅ and R²=COCl), in a suitable solvent such as tetrahydrofurantogether with a tertiary amine e.g. triethylamine, at a temperaturebetween −30° C. and +20° C., or in aqueous sodium bicarbonate solutionat 0° C. to +10° C., with amino acids, diamino acids or dipeptides, toform the corresponding, protected N-[2,3-di(benzyloxy)-benzoyl]-aminoacids, and die latter are then converted, by customary methods ofremoving the protective groups, for example by catalytic hydrogenationin ethanol, into the free catechol-substituted amino acid derivatives orpeptide derivatives (formula I, where R²=R¹¹, R¹², R¹³ or R¹⁴, whereinZ=CH₂),

or by

e. the reaction of dihydroxy- or diacyloxybenzoyl chloride (formula I,where R¹=OH or Oacyl and R²=COCl) with an oxazolidine carboxylate,obtained by known methods from serine and aldehydes, for exampleformaldehyde, in aqueous alkaline solution, with subsequentacidification in a suitable solvent, for example in ethanol or in anethanol/water mixture, at a temperature between—10° C. and +10° C., toform substituted oxazolidine,-carboxylic acid derivatives (formula I,where R²=R¹⁴ and Z=O),

or by

f. the reaction of catechol-substituted benzaldehyde (formula I, whereR²=CHO), in a suitable solvent, with O-carboxymethylhydroxylamine orsalts thereof to form the corresponding formyl-O-carboxymethyloximes(formula I, where R²=CH=NOCH₂COOH).

The compounds of formula I according to the invention in which Y inR²=an active ingredient residue which comprises a free NH or OH groupare prepared, for example, by the reaction of a compound of formula I inwhich Y in R²=OH, e.g. by the mixed anhydride method, firstly withchloroformic acid ester and a tertiary amine, e.g. triethylamine, andthen with the corresponding active ingredient which contains a free NHor OH group and which optionally contains a customary linker group, suchas residues of a diamino carboxylic acid, of a hydroxycarboxylic acid orof a diamine or diol, together with a suitable tertiary amine, e.g.triethylamine, in a suitable solvent, e.g. tetrahydrofuran.

The compounds of formula I which contain a carboxyl group may exist asfree acids, in the form of their salts or as readily cleavable esters,particularly esters which can be cleaved under physiological conditions.The compounds are purified by the usual methods known from the priorart, for example by recrystallisation or by means of chromatographicmethods.

The compounds according to the invention are effective as siderophoresfor various gram-negative bacterial strains.

Testing for siderophore efficacy was performed using various bacterialindicator mutants which only exhibit reduced growth due to lack ofsiderophores and which are capable of an increase in growth after theaddition of the test substances as substitute siderophores. In theindicator mutants, the synthesis of the respective siderophores, e.g.pyoverdin, pyochelin, enterobactin, aerobactin or yersiniabactin, or thebiosynthesis of aromatic compounds, is blocked, or there is a lack ofreceptors for enterobactin, pyochelin or pyoverdin and for otherimportant components for the bacterial transport of iron (e.g. themembrane proteins Cir, Fiu, FepA and TonB). Under conditions where thereis a lack of iron, these mutants therefore cannot grow or can only growto a very slight extent. In particular, the following indicator mutantswere used: Pseudomonas aeruginosa PAO 6609, K 407, 690; E. coli AB 2847,Salmonella typhimurium enb-7, TA 270; Klebsiella pneumoniae KN 4401;Yersinia enterocolitica WAH; Proteus mirabilis 12 (wild); Proteusvulgaris 718 (wild) and Morganella morganii SBK3 (wild). The wildstrains denoted by “wild” only possess iron absorption systems which areinadequate, which is why the addition of a siderophore results inincreased growth. The controls used were ferrioxamine E for thePseudomonas strains, ferrioxamine G and enterobactin for the Salmonellastrains, ferrichrome for the E. coli, Klebsiella and Y. enterocol.strains, and 3,4-dihydroxybenzylidene-2,4,6-trimethylaniline forMorganella morganii (see the above literature reference by R. Reissbrodtet al.).

For the E. coli mutants IR 112 and H 1728 lacking the membrane proteinsTonB or Cir and Fiu, which are important for active iron transport, allthe substances tested had no effect. This is an indication that thesubstances act purely as siderophores.

The growth areas of the indicator mutants (diameter in mm) under theeffect of the test substances are given in Tables 1-3. The annotations +and (+) relate to non-specific promotion of growth.

TABLE 1 Growth areas (in mm) of siderophore indicator strains with newsynthetic catechol derivatives Pseudomonas Salmonella Sub- aeruginosa E.coli typhimurium stance PAO AB TA No. 6609 K407 690 2847 enb-7 2700 a. 120 16 23 0 35 0 2 17 18 17 0 30 0 3 0 0 0 22 28 0 4 17 18 18 18 34 0 5 00 0 15 0 0 6 14 17 19 17 34 0 7 0 0 0 0 0 0 8 13 15 0 13 0 0 b. 9 10 0 00 10  0 0 0 22 20 11  14 0 12 0 10 0 12  14 0 12 14 0 0 13  10 11 10 1823 Control 35 35 40 23 38 20 (see text)

TABLE 2 Growth areas (in mm) of siderophore indicator strains with newsynthetic catechol derivatives Sub- Kleb- Y. enter- Proteus Proteusstance siella ocol. mir. vul. M. morg. No. KN 4401 WAH 12 718 SBK 3 a. 10 10 26 35 35 2 35 0 16 25 20 3 32 0 18 18 20 4 33 12 23 24 27 5 0 0 1921 21 6 25 23 25 25 7 23 0 20 22 22 8 20 12 20 20 20 b. 9 27 10 9 10  1826 15 16 16 11  32 10 15 20 20 12  30 25 17 20 15 13  15 10 23 23 24 14 27 10 10 12 11 15  0 12 11 Control (see text) 25 26 18 25 18

TABLE 3 Growth areas (in mm) of siderophore indicator strains with newsynthetic catechol derivatives E. M. P. aeruginosa coli S. typhimuriummorg. Substance PAO AB TA No. 6609 K407 690 2847 enb-7 2700 SBK3 c. 1620 20 20 24 25 10 17 0 26 0 12 d. 18 20 17 18 30 48 50 19 10 0 11 29 1720 10 0 18 31 12 21 14 15 15 33 36 40 22 48 40 50  0 50 50 23 14 22 2720 24 20 20 20 40 50 40 25 18 30 28 38 26 0 14 32 10 Control 35 35 40 2338 20 (see text)

Due to their properties as bacterial siderophores, the compounds ofgeneral formula I can serve as transport vehicles or penetrationaccelerators for antimicrobial antibiotics and other active ingredients,i.e. in conjugates with antibiotics or other active ingredients they canserve to transport the latter into the microbial cell via iron transportroutes and can thus increase their efficacy.

Compounds of general formula I, where Y in R²=an active ingredientresidue, possess an antibacterial efficacy, for example even in partagainst bacteria which are resistant to other β-lactams. Therefore, afew compounds of general formula I, where Y=an active ingredientresidue, e.g. substances 28-37, were tested in an agar diffusion testagainst particular bacterial strains which are in part resistant toother β-lactams (Table 4). The following strains were used: Pseudomonasaeruginosa SG 137 (carbenicillin-resistant), KW 799 WT (wild type), KW799/61 (penetration mutant, cell wall damaged, penetration made easier),ATCC 27853 (wild type), ATCC 9027 (wild type), NCTC 10662 (ATCC 25668,clinical isolate, carbenicillin-sensitive), NCTC 10701(carbenicillin-sensitive), NPS1 and Oxa6 (plasmid-coded β-lactamase); E.coli DCO (wild type), DC2 (penetration mutant, cell wall damaged,penetration made easier), Klebsiella pneumoniae ATCC 10031 (wild type),as well as SG 117; Salmonella gallinarum ATCC 9184; Stenotrophomonasmaltophilia GN 12873 (ampicillin-, azlocillin- andcarbapenem-resistant), and IMET 10402.

Surprisingly, it was found that the substances tested exhibitedoutstanding efficacy, not only for ampicillin-resistant and/orβ-lactamase inhibitor-resistant wild type strains, but that they werealso effective for two Pseudomonas strains comprising plasmid-codedβ-lactamase (NPS1, Oxa6) and multi-resistant Stenotrophomonas strains,whilst azlocillin, and in part meropenem and imipenem also, for example,were ineffective.

TABLE 4 Antibacterial efficacy of siderophore-antibiotic conjugates inthe agar diffusion test [concentration 100 μg/ml; inhibition spotdiameter in mm]. Pseudomonas aeruginosa SG KW799/ KW799/ ATCC ATCC NCTCNCTC NPS OXA Substance No. 137 WT 61 27853 9027 10662 10701 1 6Ampicillin  0  0 41  0  0  0  0  0  0 Azlocillin 17 27 40 19 15 17 27  0 0 Amoxicillin/  0  0 37  0  0  0  0  0  0 clavulanic acid imipenem 2722 30 37 19 16  0 10 12 28 20 27 31 18 17 18 19 29 28 27 33 21 23 12 1330 22 27 33 31 28 27 37 Na-31 31 31 [38] 24 32 16 25 32 Na-35 31 30 [38]22 36 25 25 29 19 Na-37 22 28 35 21 Klebsiella StenotrophomonasSalmonella pneumoniae maltophilia galinarum E. coli ATCC SG GN IMET ATCCSubstance No. DC2 DC0 10031 117 12873 10402 9184 Ampicillin 32 22 19  0 0 34 Azlocillin 33 16 15 18  0 21 21 Amoxicillin/ 29 18 21 29  0 34clavulanic acid  0  0 imipenem 27 30 21 17 22 29 24 28 27 23 17 20 24 2928 22 15 19 23 22 30 31 25 19 29 31 33 27 23 29 Na-31 38 28 21 26 29 3432 28 22 19 28 Na-35 38 24 20 23 28 33 36 19 13 17 16 24 23 Na-37 33 2722 19 16 29 [ ] at 50 μg/ml

The surprisingly good efficacy was also verified in a microdilutiontest. The minimum inhibiting concentrations (MICs) were determined forthe following bacterial strains: Pseudomonas aeruginosa NCTC 10701, NCTC10662, SG 137, ATCC 27853, KW 799 WT and KW 799/61, E. coli DCO, DC2 andATCC 25922, Serratia marcescens SG 621; Salmonella gallinarum ATCC 9184,Klebsiella pneumoniae ATCC 10031 and SG 117.

The results of the tests are given in Table 5. According to theseresults, all the siderophore-ampicillin conjugates were highly effectivecompared with azlocillin and ampicillin as the standards, particularlyagainst Pseudomonas aeruginosa SG 137, which is a germ which isparticularly resistant to carbenicillin. They were also highly effectiveagainst wild type strains of Pseudomonas, and were also effective inpart against E. coli and Serratia.

With the test germs KW 799/WT and /61 of Pseudomonas and DCO and DC2 ofE. coli, the effect of improved penetration capacity on the efficacy ofthe substances was investigated. KW 799/61 and DC2 are mutants whichpossess a more penetrable outer membrane compared with the wild types KW799/WT and DCO, respectively. For the comparison substances azlocillinand ampicillin, penetration capacities which were poor to a greater orlesser extent were determined from the considerable differences in theiractivity against the wild type and against mutants. This was in contrastto the behaviour of the conjugates, which exhibited a good penetrationcapacity.

TABLE 5 Antibacterial efficacy of siderophore-antibiotic conjugates in amicrodilution test [MIC values in μg/ml]. Pseudomonas aeruginosaKlebsiella Stenotropho- KW KW E. coli Serratia Salmonella pneumoniaemonas SG 799/ 799/ ATCC NCTC NCTC ATCC marcescens gallinarum ATCC SGmaltophilia Substance No. 137 WT 61 27853 10662 10701 DC2 DC0 25922 SG621 ATCC9184 10031 117 GN 12873 Ampicillin >100 >100 0.4 >100 >100 >1001.56 6.25 6.25 25 0.2 50 12.5 >100 Azlocillin 6.25 3.12 0.2 6.25 6.250.2 0.78 12.5 6.25 50 3.12 6.25 6.25 25-100 Amoxicillin/ >100 >100 >1006.25 3.12 >100 clavulanic acid Meropenem 0.1 0.2 <0.05 <0.05 >100 273.12 0.78 0.2 6.25 0.4 0.4 3.12 6.25 50 1.56 0.4 50 28 1.56 1.56 0.40.78 0.4 1.56 6.25 1.56 25 29 0.78 3.12 0.4 12.5 1.56 50 100 25 50 12.530 0.4 0.78 0.2 6.25 0.4 6.25 6.25 50 6.25 3.12 31 0.1 0.78 0.2 6.25 0.212.5 0.78 25 0.4 0.4 Na-31 0.1 0.78 0.78 0.2 6.25 32 1.56 1.56 0.4 1.5612.5 25 Na-35 0.1 1.56 1.58 0.2 6.25 36 0.4 3.12 6.25 25 1.56 Na-37 0.43.12 12.5 3.12 3.12 6.25

The results obtained with penetration mutants of Pseudomonas, KW 799/61,and of E. coil DC2 and wild types thereof confirmed that most of the newsubstances possessed a considerably better penetration capacity thanampicillin and azlocillin. By means of further experiments using specialE. coli mutants which lack the porins ompC and ompF, via which β-lactamsnormally enter the bacterial cell, or which lack the membrane proteintonB, which is essential for the active transport of iron, it was shownthat the siderophore-antibiotic conjugates described above are capableof utilising two penetration paths (via the porins ompC and ompF and viathe tonB iron transport path), whilst the antibiotic activity ofampicillin and azlocillin depended only on the presence of the porins.The efficacy against β-lactamase formers and against multi-resistantgerms is therefore due to a new type of mechanism for overcoming thepenetration resistance, by means of which the ratio of active ingredientto enzyme in the bacterial cell is influenced so that not all antibioticmolecules are inactivated before they reach their target.

TABLE 6 Dependence of the antibacterial activity of the new substanceson porins and tonB for mutants of E. coli (inhibition spot diameter inagar diffusion test in mm) Mutants: Substance KB5 KB4 PLB3268 BR185AB2847 No. ompC− ompF− ompF++ tonB− tonB+ 27 7.5 8.5 14 1 7 28 11 11.513 0 8.5 29 7 9.5 14 0 6 ampicillin 9.5 10 17.5 12.5 12.5 azlocillin 46.5 17.5 8 8

PLB268: ompF- was super-expressed.

Furthermore, the results of a CAS test are given in Table 7. The CAStest (chromazurol-S test) of Schwyn and Neilands (Anal. Biochem. 160, 47(1987)) is based on a colour reaction due to the dissolution leaching ofFe from the chromazurol-S complex and its binding by the catecholcompound, by means of which the property of the compound as asiderophore is detected. The CAS tests were positive for the newsubstances, whilst they were completely negative for ampicillin andazlocillin. This also verifies the surprising discovery that the newantibiotics enter the bacterial cell in an enhanced manner, namely viaan iron transport route in addition to the porin route.

TABLE 7 Complexing of Fe by the new types of antibiotic (CAS test)Substance No. CAS Test 27 ++ 28 ++ 29   +++ ampicillin − azlocillin −

On account of their properties as siderophores or as chelating agentsfor iron, compounds of general formula I, and also the salts thereofwhen acidic groups are present, and also the esters thereof which cancleave under physiological conditions, are suitable for application asdrugs for diseases which are caused by a disorder of the physiologicalmetabolism of iron. On account of their antibacterial efficacy,compounds of general formula I in which Y in R²=an active ingredientresidue, e.g. the residue of an antibiotic containing an NH or OH group,and also the salts thereof when acidic groups arc present, and theesters thereof which can cleave under physiological conditions, aresuitable as drugs for combatting bacterial infections in humans andworking animals.

Compounds of formula I can be used for said diseases either on their ownor in the form of pharmaceutical preparations with physiologicallycompatible adjuvant or carrier materials which are known in the art,wherein all customary pharmacological forms of application are possiblein principle.

EXAMPLES a. Aromatic Azomethine-carboxylic Acids

General Procedure for Examples 1, 2 and 7:

2 mmoles of the respective benzaldehyde and 2 mmoles of thecorresponding aminobenzoic acid were heated in 100 ml of dry toluene for4-5 hours under reflux, with a water separator trap fitted. The crystalswhich precipitated after cooling to room temperature or after reducingthe amount of solvent were filtered off under suction andrecrystallised.

General Procedure for Examples 3, 4, 5, 6 and 8:

2 mmoles of the respective benzaldehyde and 2 mmoles of thecorresponding aminobenzoic acid (1 mmole for diamino compounds) wereheated in 80 ml of dry ethanol for 3-4 hours under reflux. A soxhletextraction attachment filled with molecular sieve was used for bindingthe water formed during the reaction. The crystals which precipitatedafter cooling to room temperature or after reducing the amount ofsolvent were filtered off under suction and recrystallised.

Example 1 3-[3,4-di(Methoxycarbonyloxy)-benzylideneamino]-benzoic Acid(1)

Formula 1, where R¹=OCOOCH₃, R²=R⁷ in the 4-position, with R³=H, COY inthe 3-position, and X=CH, Y=OH

Substance 1 was obtained by the reaction of3,4-di(methoxycarbonyloxy)-benzaldehyde and 3-aminobenzoic acid, in ayield of 53% theoretical, as a slightly yellow solid, f.p. 198 to 199°C. (toluene).

Example 2 3-[3,4-di(Methoxycarbonyloxy)-benzylideneamino]4-hydroxybenzoic Acid (2)

Formula I, where R¹=OCOOCH₃, R²=R⁷ in the 4-position, with R³=2-OH, COYin the 5-position, X=CH, Y=OH

Substance (2) was obtained by the reaction of3,4-di(methoxycarbonyloxy)-benzaldehyde and 3-amino4-hydroxybenzoicacid, in a yield of 30% theoretical, as a slightly yellow solid with anf.p. of 221 to 223° C. (toluene).

Example 3 3-(2,3-Dihydroxy)benzylideneamino]-4-hydroxybenzoic Acid (3)

Formula I, where R¹=OH, R²=R⁷ in the 3-position, with R³=2-OH, COY inthe 5-position, X=CH, Y=OH

Substance (3) was obtained by the reaction of 2,3-dihydroxybenzaldehydeand 3-amino-4-hydroxybenzoic acid, in a yield of 59% theoretical, as redcrystals with an f.p. of 273 to 274° C. (ethanol).

Example 4 3,5-Bis-[3,4-di(methoxycarbonyloxy)-benzylideneamino]-benzoicAcid (4)

Formula I , where R¹=OCOOCH₃, R²=R⁷ in the 4-position, and where

in the 3-position, with COY in the 5-position, X=CH, Y=OH

Substance (4) was obtained by the reaction of3,4-di(methoxycarbonyloxy)benzaldehyde and 3,5-diamino-benzoic acid, ina yield of 49% theoretical, as a slightly yellow solid with an f.p. of145 to 148° C. (toluene).

Example 5 4-(2,3-Dihydroxy)benzylideneamino]-3-hydroxybenzoic Acid (5)

Formula I, where R¹=OH, R²=R⁷ in the 3-position, with R³=2-OH, COY inthe 4-position, X=CH, Y=OH

Substance (5) was obtained by the reaction of 2,3-dihydroxybenzaldehydeand 4-amino-3-hydroxybenzoic acid, in a yield of 85% theoretical, as redcrystals with an f.p. of 278 to 280° C. (ethanol).

Example 6 4-(3,4-Diacetoxybenzylideneamino)-benzoic Acid (6)

Formula I, where R¹=OCOCH₃, R²=R⁷ in the 4-position, with R³=H, COY inthe 4-position, X=CH, Y=OH

Substance (6) was obtained by the reaction of 3,4-diacetoxybenzaldehydeand 4-aminobenzoic acid, in a yield of 77% theoretical, as a yellowsolid with an f.p. of 180 to 182° C. (toluene).

Example 7 3-[(3,4-Diacetoxyphenylimino)-methyl]-4,5-dihydroxybenzoicAcid (7)

Formula I, where R¹OCOCH₃, R²=R⁸ in the 3-position, with R³=2,3-OH, COYin the 5-position, and Y=OH

Substance (7) was obtained by the reaction of3-formyl4,5-dihydroxybenzoic acid and 3,4-di(acetoxy)-aniline, in ayield of 79% theoretical, as red crystals with an f.p. of 240 to 243° C.

¹H NMR (dioxane-d₈, δ in ppm): 8.79 (s, 1 H, CH=N), 7.76 (s, 1 H, ArH),7.56 (s, 1 H, ArH), 7.28 (m, 3 H, ArH), 2.26 (s, 3 H, CH₃CO), 2.24 (s, 3H, CH₃CO).

Example 8 3-(3,4-Dihydroxyphenylazo)benzoic Acid (8)

Formula I, where R¹=OH, R²=R⁷ in the 4-position, with X=N, R³=H, COY inthe 3-position, Y=OH

3-aminobenzoic acid (612 mg, 4.5 mmoles) was diazotised with 312 mgsodium nitrite at 0° C. in 15 ml ethanol and 2.5 ml concentratedhydrochloric acid. A solution of 0.97 g (4.5 mmoles) catecholmonobenzoate in 20 ml ethanol was added at 0° C., with stirring, to thesolution of the diazonium salt. 5 ml of a 25 % Na₂CO₃ solution was thenadded to adjust the pH to 9. The reaction mixture was stirred for afurther 2 hours at 0° C., adjusted to pH 2 with HCl, and was extractedwith dichloromethane. The organic phase was washed with water and driedover sodium sulphate. The crude product obtained after removing thesolvent under vacuum was recrystallised from ethanol/water. Yield: 327mg (28 % theoretical). Red-brown crystals, f.p. 213 to 215° C.(ethanol).

¹H NMR (DMSO-d₆, δ in ppm, J in Hz): 8.28 (m, 1 H, ArH), 8.04 (dd,J=1.8, 7.8, 2 H, ArH), 7.69 (t, J=7.8, ArH), 7.42 (dd, J=2.3, 8.3 1 H,ArH), 7.36 (d, J=2.3, 1 H, ArH), 6.94 (d, J=8.3, 1 H, ArH).

b. Benzhydrazones Example 9[(3,4-Dihydroxybenzoyl)-hydrazono]-phenylacetic Acid (9)

Formula I, where R¹=OH, R²=R⁹ in the 4-position, with R⁴=COOH, R⁵=H,R¹⁵=H

1.2 g phenylglyoxylic acid (0.01 mole) in 5 ml ethanol and 1.7 g3,4-dihydroxybenzhydrazide (0.01 mole) in 5 ml of 2 N hydrochloric acidwere mixed and stirred for 1 hour at room temperature. Colourlesscrystals (ethanol). Yield 1.18 g (40% theoretical), f.p. 194 to 195° C.(decomposition).

¹H NMR (DMSO-d₆, δ in ppm): 12.8 (1H, s, NHCO), 6.86 to 7.69 (m, 8H,ArH).

Example 10 4-[(2,3-Dihydroxybenzoyl)-hydrazonomethyl]-benzoic Acid (10)

Formula I, where R¹=OH, R²=R⁹ in the 3-position, with R⁴, R¹⁵=H,R⁵=4-COOH

0.6 g (5 mmoles) terephthalaldehydic acid and 0.85 g (5 mmoles)2,3-dihydroxybenzhydrazide were boiled for 1 hour under reflux in 10 mlethanol. Colourless crystals (ethanol/water), yield 909 mg (61%theoretical), f.p. 301 to 302° C.

Example 11 4-[(3,4-Dihydroxybenzoyl)-hydrazonomethyl]-benzoic Acid (11)

Formula I, where R¹=OH, R²=R⁹ in the 4-position, with R⁴, R¹⁵=H,R⁵=4-COOH

0.6 g (5 mmoles) 4-formylbenzoic acid and 0.85 g (5 mmoles)3,4-dihydroxybenzhydrazide were boiled for 1 hour under reflux in 10 mlethanol. Colourless crystals, purified by dissolution indimethylformamide, filtration and precipitation with water, yield 889 mg(61% theoretical), f.p. 314 to 315° C.

Example 12 {[3,4-di(Methoxycarbonyloxy)-benzoyl]-hydrazono}-phenylaceticAcid (12)

Formula I, where R¹=OCOOCH₃, R²=R⁹ in the 4-position, with R⁴=COOH,R⁵=H, R¹⁵=H

300 mg (1 mmole) [(3,4-dihydroxybenzoyl)-hydrazono]-phenylacetic acid (1mmole; =the product from Example 9) were stirred for 10 minutes at 0° C.with 2 ml methyl chloroformate in 2 ml 2 N aqueous sodium hydroxidesolution and 3 ml water. The batch was then made alkaline again with 2 Naqueous sodium hydroxide solution, 1 ml methyl chloroformate was added,and the batch was stirred again for 30 minutes. It was then adjusted topH 2 with 2 N hydrochloric acid. Colourless crystals (frommethanol/water), yield 221 mg (53% theoretical), f.p. 171 to 172° C.(decomposition).

¹H NMR (DMSO-d₆, δ in ppm): 7.4-7.9 (m, 8 H, ArH), 3.87, 3.85 (2×3 HOCOCH₃).

Example 13 3-formyl-4,5-dihydroxybenzoicAcid-(2,3-dihydroxy-benzhydrazone) (13)

Formula I, where R¹OH, R²=R⁹ in the 3-position, with R⁵=5-COOH, R⁴=H,R¹⁵=3,4-OH

A mixture of 364 mg 3-formyl-4,5-dihydroxybenzoic acid, dissolved in hotwater, and 340 mg 2,3-dihydroxybenzhydrazide, dissolved in 5 ml of 2 Nhydrochloric acid, was boiled for 10 minutes with stirring. Colourlesscrystals (glacial acetic acid), yield 431 mg (65% theoretical), f.p.280-281° C. DC

¹H NMR (DMSO-D₆, δ in ppm): 8.72 (1H, s, CH=N), 6.8-7.6 (5H, ArH),7.74-7.75 (2×1H, d, J=1.6, 2- or 6 CH of benzoic acid), 7.35-7.39 and6.98-7.02 (2×1H, d, J=8.4 and 6 CH of benzhydrazone), 6.74-6.82 (1H, t,J=8.5, CH of benzhydrazone).

Example 144-{[3,4-di-(Methoxycarbonyloxy)-benzoyl]-hydrazono-methyl}-benzoic Acid(14)

Formula I, where R¹=OCOOCH₃, R²=R⁹ in the 4-position, with R⁵=4-COOH,R⁴=H, R¹⁵=H,

300 mg(1 mmole) 4-[(3,4-dihydroxybenzoyl)-hydrazono-methyl]-benzoic acid(=the product from Example 11) were dissolved in 1 ml of 2 N aqueoussodium hydroxide solution and 3 ml water, and the solution was cooled to0° C. and treated with 2 ml methyl chloroformate with stirring. Themixture was stirred for a further 30 minutes with additional cooling andwas then adjusted to pH 3 with HCl. Colourless crystals (ethanol/water),yield 205 mg (49% theoretical), f.p. 184 to 187° C. (decomposition).

1H NMR (DMSO-D₆, δ in ppm): 8.50 (s, 1H, CH=N), 7.62-8.04 (m 7H, ArH),3.88 (s, 6H, 2×OCOOCH₃).

Example 156-[(3,4-Dihydroxybenzoyl)-hydrazonomethyl]-2,3-dihydroxybenzoic Acid(15)

Formula I, where R¹=OH, R²=R⁹ in the 4-position, with R⁵=2-COOH, R⁴=H,R¹⁵=3,4-OH

A mixture of 182 mg (1 mmole) 6-formyl-2,3-dihydroxybenzoic acid,dissolved in hot water, and 168 mg (1 mmole) 3,4-dihydroxybenzhydrazide,dissolved in 5 ml of 2 N hydrochloric acid, was boiled for 10 minuteswith stirring. Pale yellow crystals (water). Yield 215 mg (65%theoretical), f.p. 252° C.

1H NMR (DMSO-D₆, δ in ppm): 8.53 (s, 1 H, CH=N), 6.8-7.3 (m, 5 H, ArH).

c. Aminobenzoic Acid Derivatives Example 162-(2,3-Diacetoxy-benzoylamino)-benzoic Acid (16)

Formula I, where R¹=OCOCH₃, R²=R¹⁰ in the 3-position, with COY in the2-position, Y=OH, R¹⁸-R²⁰=H

Method 1:

1.50 g (0.011 mole) anthranilic acid were slurried in 100 ml of 0.5 MNaHCO₃ solution, and were treated at 0 to 10° C., in an ultrasonic bathwith stirring, with 2.56 g (0.01 mole) 2.3-diacetoxybenzoyl chloride in8 ml tetrahydrofuran. The turbid solution which formed after 45 minuteswas filtered and carefully acidified with concentrated hydrochloricacid. The colourless crystals obtained were washed with a little ethylacetate and dried under vacuum. f.p. 203 to 204° C., yield 2.7 g (75%theoretical).

¹H NMR (DMSO-D₆, δ in ppm): 11.8 (s, 1H, NHCO), 7.2-8.5 (m, 7H, ArH),2.15 and 2.25 (s, 2×3 H, OCOCH₃).

Method 2:

2.57 g 2,3-diacetoxybenzoyl chloride in 50 ml tetrahydrofuran were addedat 0° C. to 1.4 g (0.01 mole) anthranilic acid in 100 ml sodiumbicarbonate solution. The mixture was reacted for 1 hour at 0-10° C. andthen for 30 minutes in an ultrasonic bath at room temperature. It wasthen partially concentrated and was carefully acidified with 2 Nhydrochloric acid. Pale yellow crystals (ethyl acetate), yield 2.3 g(64% theoretical), f.p. 198 to 200° C.

Example 16a 4-[(2,3-Dimethoxycarbonyloxy-benzoyl)-methylamino]benzoicAcid

Formula I, where R¹=OCOOCH₃, R²=R¹⁰ in the 3-position, R¹⁹=CH₃,R¹⁸=R²⁰=H, COY in the 4-position, Y=OH

Using method 1, the title compound was obtained analogously from4-methylaminobenzoic acid and 2,3-di-(methoxy-carbonyloxy)-benzoylchloride, in a yield of 65% theoretical.

Example 16b 3,5-bis-(2,3-Diacetoxybenzoylamino)-benzoic Acid

Formula I, where R¹=OCOCH₃, R²=R¹⁰ in the 3-position, R¹⁸=R¹⁹=H,R²⁰=(2,3-diacetoxy)-benzoylamino in the 5-position with COY in the3-position, Y=OH

Using method 1, the title compound was obtained analogously from3,5-diaminobenzoic acid and 2,3-diacetoxy)-benzoyl chloride, in a yieldof 55% theoretical.

Example 17 4-(2,3-Diacetoxy-benzoylamino)benzoic Acid (17)

Formula I, where R¹=OCOCH₃, R²=R¹⁰ in the 3-position, with COY in the4-position, Y=OH, R¹⁸-R²⁰=H

257 mg 2,3-diacetoxybenzoyl chloride in 5 ml tetrahydrofuran were addedto 137 mg (1 mmole) 4-aminobenzoic acid and 0.14 ml triethylamine in 10ml tetrahydrofuran. The mixture was stirred for 30 minutes at 20° C. andthen for 30 minutes at room temperature. It was then partiallyconcentrated and was carefully acidified with 2 N hydrochloric acid.Pale yellow crystals (ethyl acetate), yield 215 mg (60% theoretical).f.p. 134-135° C.

¹H NMR (DMSO-D₆, δ in ppm): 7.3-7.9 (m 7 H, ArH), 2.1 and 2.3 (s, 2×3 H,OCOCH₃).

d. Amino Acid Derivatives

General Procedure for Examples 19, 20, 22, 23 and 25:

1st Step (acylation):

2.5 mmoles of the respective amino acid or dipeptide (present in itsfree form or as the hydrochloride or acetate) were dissolved in 10 ml ofaqueous NaOH solution (2.5 mmoles NaOH for alanine, 7.6 mmoles fordiamino acids, 11.5 mmoles for the dipeptide). A solution of2,3-di(benzyloxy)-benzoyl chloride (2.5 mmoles for alanine, 5.1 mmolesfor diamino acids, 9 mmoles for the dipeptide) in 10 ml THF was slowlyadded drop-wise at 0° C. with stirring, followed by warming to 20 to 25°C. when the addition was complete. The reaction mixture was stirred for4 hours at this temperature and was subsequently adjusted to pH 2 with 2M HCl. The reaction mixture was extracted with ethyl acetate, theorganic phase was washed with saturated aqueous sodium chloride solutionand dried over sodium sulphate, and the solvent was removed undervacuum. The crude product could be purified by recrystallisation(ethanol/water in the case of the alanine derivative) or by columnchromatography (silica gel 60, Merck, eluent:chloroform:ethylacetate:glacial acetic acid=30:10:1 or ethyl acetate:toluene:glacialacetic acid=10:10:2).

2nd Step (debenzylation):

500 mg of the product formed in the 1st step were dissolved in a mixtureof 9 ml ethanol and 1 ml glacial acetic acid, treated with 50 mgpalladium on activated carbon (10% Pd) and stirred at 20° C. undernormal pressure in a hydrogen atmosphere until the requisitestoichiometric amount of hydrogen had been absorbed (normally 4 to 6hours). The reaction mixture was then filtered through celite, thesolvent was distilled off, and the residue was dried under vacuum.

General Procedure for Examples 21, 24 and 26:

1st Step (acylation):

2 mmoles of the amino acid benzyl ester hydrochloride or tosylate weredissolved in 10 ml dichloromethane and treated with 4 mmolestriethylamine (8 mmoles in the case of diamino acid derivatives). Asolution of 2 mmoles of 2,3-diacetoxybenzoyl chloride (4 mmoles fordiamino acid derivatives) in 10 ml dichloromethane was slowly addeddrop-wise at −30° C. The reaction mixture was then stirred for a further1 hour at −30° C. and for 1 hour at 20-25° C. The reaction solution waswashed in succession with 1 M hydrochloric acid, saturated aqueoussodium hydrogen carbonate solution and saturated aqueous sodium chloridesolution. The organic phase was dried over sodium sulphate, the solventwas removed under vacuum, and the residue was purified byrecrystallisation from ethanol or toluene (in the case of the serinederivative) or by column chromatography (lysine derivative: silica gel60, mobile phase:ethyl acetate:toluene=2:1).

2nd Step (cleavage of benzyl ester):

1 g of the product formed in the 1st step was dissolved in a mixture of20 ml ethanol and 1 ml glacial acetic acid, treated with 100 mgpalladium on activated carbon (10% Pd) and stirred for 2 hours at 20° C.under normal pressure in a hydrogen atmosphere. The reaction product wasfiltered through celite and the solvent was removed under vacuum. Theresidue was taken up in ethyl acetate and washed with saturated aqueoussodium chloride solution, and the organic phase was dried over sodiumsulphate. After removing the solvent under vacuum, the productcrystallised out or was purified by recrystallisation.

Example 18 L-2,5-bis-(2,3-Dihydroxybenzoylamino)-pentanoic Acid (18)

Formula I, where R¹=OH, R²=R¹¹ in the 3-position, and with R⁶=R¹², n=3,R¹⁵=2,3-OH, Y=OH, L-form,

Substance (18) was obtained from L-ornithine monohydrochloride, in ayield of 66% theoretical, as a white solid.

¹H NMR (DMSO-d₆, δ in ppm): 7.41 (dd, 1 H, ArH), 7.27 (dd, 1 H, ArH),6.92 (m, 2 H, ArH), 6.69 (m, 2 H, ArH), 4.46 (m, 1 H, CH-N), 3.36 (m, 2H, CH₂), 1.84 (m, 2 H, CH₂), 1.65 (m, 2 H, CH₂).

Example 19 L-2-(2,3-Dihydroxybenzoylamino)-propionic Acid (19)

Formula I, where R¹=OH, R²=R¹¹ in the 3-position, and with R⁶=CH₃, Y=OH,L-form

Substance (19) was obtained from L-alanine, in a yield of 88%theoretical, as a white solid.

¹H NMR (DMSO-d₆, δ in ppm): 7.40 (dd, 1 H, ArH), 6.94 (dd, 1 H, ArH),6.72 (dd, 1 H, ArH), 4.44 (m, 1 H, CH), 1.42 (d, 3 H, CH₃).

Example 20 L-2-(2,3-Diacetoxybenzoylamino)-propionic Acid (20)

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, with R⁶=CH₃, Y=OH,L-form

Substance (20) was obtained from L-alanine benzyl ester hydrochloride,in a yield of 75% theoretical, as colourless crystals with an f.p. of109° to 111° C.

¹H NMR (DMSO-d₆, δ in ppm): 7.50 (dd, 1 H, ArH), 7.39 (m, 2 H, ArH),4.33 (m, 1 H, CH), 2.29 (s, 3 H, CH₃CO), 2.23 (s, 3 H, CH₃CO), 1.34 (d,3 H, CH₃).

Example 21 L-2,4-Bis-(2,3-dihydroxybenzoylamino)-butyric Acid (21)

Formula I, where R¹=OH, R²=R¹¹ in the 3-position, with R⁶=R¹², n=2,R¹⁵=2,3-OH, Y=OH, L-form

Substance (21) was obtained from L-2,4-diaminobutyric aciddihydrochloride, in a yield of 81% theoretical, as a grey-white solid.

¹H NMR (DMSO-d₆, δ in ppm): 7.42 (dd, 1 H, ArH), 7.27 (dd, 1 H, ArH),6.94 (m, 2 H, ArH), 6.72 (m, 2 H, ArH), 4.48 (m, 1 H, CH-N), 3.42 (m, 2H, CH₂-N), 2.21 (m, 1 H, CH₂), 2.05 (m, 1 H, CH₂).

Example 22L-3-[2,6-Bis-(2,3-dihydroxybenzoylamino)]-hexanoyl-amino]-2-(2,3-dihydroxybenzoylamino)-propionicAcid (22)

Formula I, where R¹=OH, R²=R¹¹ in the 3-position, with R⁶=R¹³, n₁=1,n₂=4, R¹⁵=2,3-OH, Y=OH, L-form

Substance (22) was obtained fromL-2-amino-3-(2,6-diamino-hexanylamino)-propionic acid, in a yield of 70%theoretical, as a white solid.

¹H NMR (DMSO-d₆, δ in ppm): 7.42 (dd, 1 H, ArH), 7.26 (m, 2 H, ArH),6.91 (m, 3 H, ArH), 6.69 (m, 2 H, ArH), 6.67 (m, 3 H, ArH), 4.55-4.28(m, 3 H, CH-N, CH₂-N), 4.10-3.09 (m, 3 H, CH₂-N), 1.72 (m, 2 H, CH₂),1.51-1.28 (m, 4 H, CH₂).

Example 23 L-2-[2,3-Diacetoxybenzoylamino)-3-hydroxypropionic Acid (23)

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, with R⁶=CH₂, OH,Y=OH, L-form

Substance (23) was obtained from L-serine benzyl ester hydrochloride, ina yield of 53% theoretical, as colourless needles, with an f.p. of 165to 168° C. (acetone/hexane).

¹H NMR (DMSO-d₆, δ in ppm): 7.59 (dd, 1 H, ArH), 7.42 (m, 2 H, ArH),4.42 (m, 1 H, CH), 3.76 (m, 1 H, CH₂O), 2.29 (s, 3 H, CH₃CO), 2.28 (s, 3H, CH₃CO).

Example 23a L-3-Benzyloxy-2-(2,3-diacetoxybenzoylamino)-propionic Acid

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, R⁶=CH₂OCH₂ (C₆H₅),Y=OH, L-form.

This substance was obtained in the form of white crystals fromO-benzyl-L-serine benzyl ester, analogously to substance 23, but withthe use of palladium on activated carbon (10% Pd) and cyclohexadiene instep 2.

Example 24 D-2,5-Bis-(2,3-dihydroxybenzoylamino)-pentanoic Acid (24)

Formula I, where R¹=OH, R²=R¹¹ in the 3-position, with R⁶=R¹², n=3,R¹⁵=2,3-OH, Y=OH, D-form

Substance (24) was obtained from D-ornithine monohydrochloride, in ayield of 66% theoretical, as a white solid.

¹H NMR (DMSO-d₆, δ in ppm): 7.41 (dd, 1 H, ArH), 7.27 (dd, 1 H, ArH),6.93 (m, 2 H, ArH), 6.70 (m, 2 H, ArH), 4.46 (m, 1 H, CH-N), 3.33 (m, 2H, CH₂-N), 1.84 (m, 2 H, CH₂), 1.64 (m, 2 H, CH₂).

Example 25 1L-2,6-Bis-(2,3-diacetoxybenzoylamino)-hexanoic Acid (25)

Formula I, R¹=OCOCH₃, R²=R¹¹ in the 3-position, with R⁶=R¹², n=4,R¹⁵=2,3-OCOCH₃, Y=OH, L-form

Substance (25) was obtained from L-lysine benzyl ester ditosylate, in ayield of 71% theoretical, as a white solid.

¹H NMR (CDCl₃, δ in ppm, J in Hz): 7.65 (dd, J=1.9, 7.4, 1 H, ArH), 7.47(dd, J=2.3, 7.2, 1 H, Ar-H), 7.32-7.22 (m, J=1.8, 7.3, 5 H, ArH, CONH),6.59 (t, J=5.7, 1 H, CONH), 4.75 (m, J=5.2, 7.2, 1 H, CH-N), 3.38 (m, 2H, CH₂-N), 2.34 (s, 3 H, CH₃CO), 2.30 (s, 3 H, CH₃CO), 2.30 (s, 3 H,CH₃CO), 2.29 (s, 3 H, CH₃CO), 1.99 (m, 1 H, CH₂), 1.85 (m, 1 H, CH₂),1.60 (m, 2 H, CH₂), 1.28 (m, 2 H, CH₂).

e. Oxazolidine-carboxylic Acid Residues Example 26(S)-3-(2,3-diacetoxybenzoyl)-oxazolidine-4-carboxylic Acid (26)

Formula I, where R¹=OCOCH₃, R²=R¹⁴ in the 3-position, with Z=O,R¹⁶=R¹⁷=H, Y=OH, S-form

L-serine (105 mg, 1 mmole) was dissolved in 0.5 ml of 2 M aqueous sodiumhydroxide solution and was treated at 0° C. with 0.1 ml of aqueousformaldehyde solution (36.5%). The reaction mixture was allowed to standfor 24 hours at 0° C.; 84 mg (1 mmole) sodium hydrogen carbonate and 1ml acetone were then added and the mixture was cooled to −5° C. 257 mg(1 mmole) 2,3-di(acetoxy)benzoyl chloride were then added in portions,with stirring. After stirring for 1 hour at −5° C. to 0° C., thereaction mixture was diluted with 10 ml water and extracted with diethylether. The aqueous phase was acidified to pH 2-3 with 1 M hydrochloricacid and extracted with diethyl ether again (3×20 ml). The organic phasewas dried over sodium sulphate and the solvent was removed under vacuum.The product obtained was dried under vacuum. Yield: 263 mg (78%theoretical). White foam.

¹H NMR (CDCl₃, δ in ppm): 7.35-7.27 (m, 3 H, ArH), 4.90 (s, 2 H,O-CH₂-N), 4.82 (m, 1 H, CH), 4.39 (m, 1 H, CH₂), 4.28 (m, 1 H, CH₂),2.33 (s, 3 H, CH₃), 2.31 (s, 3 H, CH₃).

f. Conjugates With Antibiotics Example 27N-[L-2-(2,3-Diacetoxybenzoylamino)-propionyl]-ampicillin

(IUPAC nomenclature: 6-{2-[2-(2,3-diacetoxybenzoylamino)-propionylamino]-2-phenyl-acetylamino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid) (27)

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, with R⁶=CH₃(L-form), Y=N-ampicillino

500 mg (1.62 mmoles) L-2-(2,3-diacetoxybenzoylamino)-propionic acid(substance 20) were dissolved in 15 ml tetrahydrofuran, and 0.18 ml(1.62 mmoles) N-methylmorpholine followed by 0.21 ml (1.62 mmoles)isobutyl chloroformate were added drop-wise thereto at −20° C. withstirring. After stirring for 1 hour, a solution of 565 mg (1.62 mmoles)ampicillin trihydrate in 5 ml of 80% tetrahydrofuran, which was cooledto 0° C., was added drop-wise. The mixture was stirred for 1 hour at−20° C. and for 1 hour at room temperature and was then concentratedunder vacuum. Water and ethyl acetate were then added and the mixturewas carefully acidified to pH 2 with 1 M hydrochloric acid. The mixturewas thoroughly and rapidly shaken until everything had dissolved, andthe ethyl acetate phase was separated, washed until neutral with aqueouscommon salt solution, and dried over sodium sulphate. Afterconcentration under vacuum, precipitation was effected with petroleumether. Yield of crude product: 1 g; purity according to HPLC (Europher100-7): about 75%). Purification was effected by way of preparative HPLC(RP₁₈, acetonitrile/water=40:60+0.50 % trifluoroacetic acid, flow rate10 ml/minute). The fractions which contained the product wereimmediately extracted with ethyl acetate, and the organic phase waswashed with water, dried, concentrated and the product was precipitatedwith petroleum ether. Purity according to HPLC: 95%.

¹H NMR (300 MHz, CDCl₃, δ in ppm, J in Hz): 7.63 (dd, J=1.9, 7.4, ArH),7.33-7.19 (m, 7H, Ar-H), 5.61 (m, J=4.1, 2 H, CH-N), 5.42 (d, J=4.1, 1H, CH-S), 5.00 (m, J=7.3, 1 H, CH-Me), 4.29 (s, 1H, CH-COO), 2.30 (s, 3H, CH₃CO), 2.27 (s, 3 H, CH₃CO), 1.43 (s, 3 H, CH₃), 1.40 (d, J=7.1, 3H, CH₃), 1.37 (s, 3H, CH₃).

Example 28 N-[2-(2,3-Diacetoxybenzoylamino)-benzoyl]-ampicillin

(IUPAC nomenclature:6-{2-[2-(2,3-diacetoxybenzoylamino)-benzoylamino]-2-phenyl-acetylamino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid) (28)

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, R¹⁸-R²⁰=H, withCOY in the 2-position, Y=N-ampicillino

0.11 ml (1 mmole) N-methylmorpholine and a catalytic amount of4-dimethylaminopyridine were first added at −20° C. to a solution of 357mg (1 mmole) 2-(2,3-diacetoxybenzoylamino)-benzoic acid (substance 17)in 5 ml of absolute tetrahydrofuran, followed by the addition of 126 μlof isobutyl chloroformate with stirring. The mixture was stirred for 1hour at −20° C., and then a solution of 371 mg (1 mmole) of the sodiumsalt of ampicillin in 3 ml of 80% tetrahydrofuran was added in portions.The mixture was stirred for 1 hour at −20° C. and for 2 hours at roomtemperature. The solvent was then distilled off under vacuum, and 20 mlwater and 20 ml ethyl acetate were added to the residue. The mixture wascarefully acidified to pH 3 with 1 M hydrochloric acid and wasthoroughly shaken. The organic phase was separated, washed three timeswith aqueous common salt solution, and dried over sodium sulphate. Afterconcentration, precipitation was effected with petroleum ether. Yield ofcrude product: 420 mg.

The crude product was separated by means of preparative HPLC (Nucleosil7 C 18, Macherey & Nagel, mobile phase 50/50 acetonitrile/water+0.05%trifluoroacetic acid

2nd Fraction

N-(2-(2,3-diacetoxybenzoylamino)-benzoyl-ampicillin

Yield from 420 mg of crude product: about 90 mg, HPLC (Europher 100 C18-7, mobile phase 60/40 acetonitrile/water+0.05% trifluoroacetic acid).

¹H NMR (DMSO-d₆, δ in ppm): 7.3-8.4 (m 12 H, ArH), 5.9 (d, 1 H, J=7.7Hz, CH), 5.53 (q, 1 H, CH), 5.42 (d, 1 H, J=4.0 Hz, CH), 4.2 (s, 1 H,CH), 2.32, 2.24 (s, 2×3 H, CH₃), 1.40, 1.50 (s, 2×3 H, CH₃).

Example 29 N-(4-(2,3-Diacetoxybenzoylamino)-benzoyl-ampicillin (29)

Formula I, where R¹=OCOCH₃, R²=R¹⁰ in the 3-position, R¹⁸-R²⁰=H, withCOY in the 4-position, Y=N-ampicillino

Substance (29) was obtained, analogously to substance 28, from4-(2,3-diacetoxybenzoylamino)-benzoic acid (substance 17) and the sodiumsalt of ampicillin. The crude product was separated by means ofpreparative HPLC (Nucleosil 7 C 18, Macherey & Nagel, mobile phase 50/50acetonitrile/water+0.05% trifluoroacetic acid.

¹H NMR (DMSO-d₆, δ in ppm): 7.3-7.9 (m 12 H, ArH), 5.9 (d, 1 H, α-CH),5.53 (q, 1 H, 6-CH), 5.41 (d, 1 H, 7-CH), 4.2 (s, 1 H, 3-CH), 2.20, 2.28(s, 2×3H, CH₃), 1.40, 1.52 (s, 2×3 H, CH₃).

Example 30(S)-N-[3-(2,3-di-(Methoxycarbonyloxy)-benzoyl]-oxazolidin-4-oyl]-ampicillin

(IUPAC nomenclature:(S)-6-(2-{3-(2,3-di-(methoxycarbonyloxy)-benzoyl)-oxazolidine-4-carbonyl]-amino)-2-phenylacetylamino)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid) (30)

Formula I, where R¹=OCOOCH₃, R²=R¹⁴ in the 3-position, with Z=O, R¹⁶,R¹⁷=H, (S-form), Y=N-ampicillino

Substance 30 was obtained, analogously to substance 28, from(S-3-(2,3-di-(methoxycarbonyloxy-benzoyl)-oxazolidine4-carboxylic acidand ampicillin trihydrate.

Example 31 N-[L-2,6-bis-(2,3-diacetoxybenzoylamino)-hexanoyl]-ampicillin

(IUPAC nomenclature:6-{L-2-(2,6-bis-(2,3-diacetoxy-benzoylamino)-hexanoylamino]-2-phenylacetylamino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid) (31)

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, with R⁶=R¹², n=4,R¹⁵=2,3-OCOCH₃, (L-form), Y=N-ampicillino

Substance 31 was obtained, analogously to substance 28, fromL,2,6-bis-(2,3-diacetoxybenzoylamino)-hexanoic acid (substance 25) andampicillin trihydrate.

Example 32N-[L-3-Acetoxy-2-(2,3-diacetoxybenzoylamino)-propionyl]-ampicillin

(IUPAC nomenclature:6-{L-2-[3-acetoxy-2-(2,3-diacetoxybenzoylamino)-propionylamino]-2-phenylacetylamino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid) (32)

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, with R⁶=CH₂COOCH₃(L-form), Y=N-ampicillino

Substance 32 was obtained, analogously to substance 27, fromL-3-acetoxy-2-(2,3-diacetoxybenzoylamino)-propionic acid and ampicillintrihydrate. The purity of the crude product as determined by HPLC(Nucleosil 7 C 18, Macherey & Nagel, mobile phase 40/60acetonitrile/water+0.05% trifluoroacetic acid, flow rate: 1 ml/min) was85%.

Example 33 N-[L-2-(2,3-diacetoxybenzoylamino)-propionyl]-cephadroxil

(IUPAC nomenclature:7-{L-2-[2-(2,3-diacetoxybenzoylamino)-propionylamino]2-(4-hydroxyphenyl)-acetylamino-3-methyl-8-oxo-5-thia-1-aza-bicyclo[4.2.]oct-2-ene-2-carboxylicacid) (33)

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, with R⁶=CH₃(L-form), Y=N-cephadroxil (33)

Substance 33 was obtained, analogously to substance 28, fromL-2-(2,3-diacetoxybenzoylamino)-propionic acid (substance 20) andcephadroxil. Purification was effected by means of preparative HPLC(RP18, 30:70 acetonitrile/water+0.1% trifluoroacetic acid).

Compound (33) which was obtained had a purity of 91%.

Example 34N-[L-3-Benzyloxy-2-(2,3-diacetoxy-benzoylamino)-propionyl]-ampicillin

Formula I, where with R¹=OCOCH₃, R²=R¹¹ in the 3-position,R⁶=CH₂OCH₂(C₆H₅), L-Form, Y=N-ampicillino

This substance was obtained, analogously to substance 28, fromL-3-benzyloxy-2-(2,3-diacetoxy-benzoylamino)-propionic acid (substance23a) and ampicillin trihydrate.

Example 35N-[L-2,6-bis-(2,3-diacetoxy-benzoylamino)-hexanoyl)-amoxicillin

Formula I, where R¹=OCOCH₃, R²=R¹¹ in the 3-position, R⁶=R¹², n=4,R¹⁵=2,3-OCOCH₃, L-Form, Y=N-amoxicillino

This substance was obtained, analogously to substance 28 fromL-2,6-bis-(2,3-diacetoxybenzoylamino)-hexanoic acid (substance 25) andamoxicillin trihydrate, in a yield of 84% theoretical.

Example 36 N-[3,5-bis-(2,3-diacetoxy-benzoylamino)-benzoyl]-ampicillin

Formula I, where R¹=OCOCH₃, R²=R¹⁰ the 3-position, R¹⁸=R¹⁹=H.R²⁰=(2,3-diacetoxy)-benzoylamino in the 5-position, with COY in the3-position, Y=N-ampicillino

This substance was obtained, analogously to substance 28, from3,5-bis-(2,3-diacetoxy-benzoylamino)-benzoic acid (substance 16b) andthe sodium salt of ampicillin.

Example 37N-{4-[(2,3-di-methoxycarbonyloxy-benzoyl)-methyl-amino]-benzoyl}-ampicillin

Formula I, where R¹=OCOOCH₃, R²=R¹⁰ in the 3-position, R¹⁹=CH₃,R¹⁸=R²⁰=H, COY in the 4-position, Y=N-ampicillino

A solution of 280 mg4-[(2,3-di-methoxycarbonyloxy-benzoyl)-methyl-amino]-benzoyl chloride(prepared from4-[(2,3-di-methoxycarbonyloxy-benzoyl)-methyl-amino]-benzoicacid=substance 16a and phosphorus pentachloride) was added drop-wise at−5° C., with stirring, to a solution of 245 mg of the sodium salt ofampicillin in 5 ml of aqueous tetrahydrofuran (80% THF). The mixture wasstirred for 1 hour at 0° C. and for 1 hour at 20° C. and was thenconcentrated under vacuum. The residue was then brought to pH 3 with 1 Nhydrochloric acid and was extracted with ethyl acetate. The extractswere washed with aqueous sodium chloride solution and were dried oversodium sulphate. After extensive concentration by evaporation, the batchwas treated with petroleum ether. 425 mg (88% theoretical) of the titlecompound were thereby precipitated in the form of a white powder.

Na Salts of Substances 27, 28, 30, 31, 35 and 37

The sodium salts of the aforementioned substances could be obtained bydie following general procedure:

A solution of 1.1 g of the acid in 5 ml ethyl acetate was treated with asolution of 0.5 g sodium 2-ethyl-hexanoate in 3 ml ethyl acetate and themixture was diluted with 30 ml petroleum ether (boiling point 40-65°C.). The sodium salts which were thereby precipitated were isolated,dried under vacuum and purified by means of preparative HPLC in an RP 18column. Yields: 50-80%

What is claimed is:
 1. Catechol derivatives of general formula I

wherein the R¹ radicals denote O-acyl, and R² represents the followinggroups in the 3- and/or 4-position: amino acid residues:

Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion, an ammoniumion or a substituted ammonium ion, or Y=a residue of an antibacterialactive ingredient which contains an OH or NH group, R⁶=alkyl, C₁-C₅hydroxyalkyl, or alkoxyalkyl, acyloxyalkyl, arylalkoxyalkyl, or

R¹⁵ represents, identically to or independently of each other, H, OH,O-acyl, n is an integer between 1 and 5 when R¹⁵ is H and/or O-acyl or

R¹⁵=radicals which, identically to or independently of each other,denote H, OH, O-acyl, n₁ and n₂ represent an integer between 1 and
 5. 2.Compounds of formula I according to claim 1, wherein R²=R¹¹, with Y=OH,and R⁶=a C₁-C₄ alkyl.
 3. Compounds of formula I according to claim 1,wherein R²=R¹¹, with Y=OH, R⁶=R¹², and with R¹⁵=O-acyl and n=1-5. 4.Compounds of formula I according to claim 1, wherein R²=R¹¹, with Y=OH,R⁶=R¹³ with R¹⁵=O-acyl and n₁ and n₂ denote an integer between 1 and 4.5. Compounds of formula I according to claim 1, wherein Y is the residueof a cephalosporin.
 6. Compounds of formula I according to claim 1,wherein Y is the residue of a penicillin.
 7. Compounds of formula Iaccording to claim 1, wherein Y is an ampicillin residue.
 8. Compoundsof formula I according to claim 1, wherein Y is an amoxicillin residue.9. Compounds of formula I according to claim 1, wherein Y is atetracycline residue comprising an NH or OH group.
 10. Compounds offormula I according to claim 1, wherein Y is the residue of macrolidecomprising an NH or OH group.
 11. Compounds of formula I according toclaim 1, wherein Y is the residue of a quinolone comprising an NH or OHgroup.
 12. Compounds of formula I according to claim 1, wherein Y is theresidue of a carbapenem comprising an NH or OH group.
 13. Compounds offormula I according to claim 1, wherein R⁶ is a C₁-C₅ alkyl or C₁-C₅hydroxyalkyl.
 14. The compound of general formula I according to claim1, wherein the compound is L-2-(2,3-diacetoxybenzoylamino)-propionicacid.
 15. The compound of general formula I according to claim 1,wherein the compound is L-2,6-bis-(2,3-diacetoxybenzoylamino)-hexanoicacid.
 16. The compound of general formula I according to claim 1,wherein the compound isN-[L-2-(2,3-diacetoxybenzoylamino)-propionyl]-ampicillin.
 17. Thecompound of general formula I according to claim 1, wherein the compoundisN-[L-3-acetoxybenzoy-2-(2,3diacetoxy-benzoyl-amino)-propionyl]-ampicillin.18. A pharmaceutical composition comprising a compound of formula Iaccording to claim 1 and a pharmaceutically acceptable excipient orcarrier.
 19. A method of treating a patient suffering from a bacterialinfection comprising administering to said patient an effectiveanti-bacterial amount of a compound of formula I according to claim 1.